Program Director/Principal Investigator (Last, First, Middle): The overall goal of this Project is to identify cellular proteins that are required for Ebola virus replication. Ebola viruses pose significant risks to human health and national security. The 2014 outbreak in Western Africa infected more than 28,000 people and caused more than 11,000 deaths (per WHO statistics). Although vaccine candidates are showing promise in clinical trials, no clinically approved drugs are available and basic information about Ebola virus replication is lacking. Like all viruses, Ebola virus depends on cellular proteins for its replication. This dependence creates a potential ?Achilles heel? that may be exploited to develop new approaches to treat Ebola virus infections. By studying Ebola virus-host cell interactions, we gain insight into the mechanisms of action of viral proteins and the strategies that the virus uses to interface with its host. However, relatively few host factors of Ebola virus replication have been reported and no systematic, genome- wide analyses have been performed. To address this gap, we will apply complementary technologies to develop high quality maps of Ebola virus-host cell interactions. In Aim 1 we perform a high content, genome- wide RNAi screen under BSL4 conditions with wildtype Ebola virus to identify novel host factors required for Ebola virus replication. The capacity to perform such a screen is unique to the research team. Since the host factors identified in the siRNA screen could act directly or indirectly to affect Ebola virus replication, in Aim 2 we propose to identify cellular proteins that bind to Ebola virus replication proteins. Complementary protein- protein interaction discovery technologies (yeast two-hybrid assay and co-affinity purification plus mass spectrometry) are used to develop a more comprehensive Ebola virus-host cell protein interaction network than could be achieved with any single approach. Since the sequences of Ebola virus strains differ between outbreaks and the impact of these genetic changes on host cell interactions is not known, we systematically compare host cell binding partners of a 2014 outbreak strain to those of Ebola Zaire (1976). Host cell factors from Aims 1 and 2 are integrated with existing large-scale data sets in Aim 3 to develop strain-specific Ebola virus-host cell protein interaction networks. To create a more complete understanding of regulatory effects of the virus-host interactions, we will enrich the network in indirect transcriptional interactions using high- throughput RNA-seq profiling of a subset of host factors selected from Aims 1 and 2. Our research team is well qualified to accomplish the goals of this Project, having expertise in high-throughput screening of BSL4 viruses, virus-host cell protein interactions, RNA-Seq profiling, and network-based bioinformatic analyses. Host factors will be tested for their effects on virus infection in Projects 1 and 3, and characterized by biophysical and structural methods in Core B and Project 2. Together these studies will develop the most comprehensive analysis of Ebola virus-host cell interactions to date. The data is integral to the mission of this Program and is expected to stimulate hypothesis-driven experiments in broader Ebola virus research community. 0925-0001/0002 (Rev. 08/12) Page Continuation Format Page